Patellofemoral pain (PFP) is commonly caused by abnormal pressure on the knee due to excessive load while standing, squatting, or going up or down stairs. To better understand the pathophysiology of PFP, we conducted a noninvasive patellar tracking study using a C-arm computed tomography (CT) scanner to assess the non-weight-bearing condition at 0o knee flexion (NWB0o) in supine, weight-bearing at 0o (WB0o) when upright, and at 30o (WB30o) in a squat. Three-dimensional (3D) CT images were obtained from patients with PFP (12 women, 6 men; mean age, 31 ± 9 years; mean weight, 68 ± 9 kg) and control subjects (8 women, 10 men; mean age, 39 ± 15 years; mean weight, 71 ± 13 kg). Six 3D-land-marks on the patella and femur were used to establish a joint coordinate system (JCS) and kinematic degrees of freedom (DoF) values on the JCS were obtained: patellar tilt (PT, o), patellar flexion (PF, o), patellar rotation (PR, o), patellar lateral-medial shift (PTx, mm), patellar proximal-distal shift (PTy, mm), and patellar anterior-posterior shift (PTz, mm). Tests for statistical significance (p < 0.05) showed that the PF during WB30o, the PTy during NWB0o, and the PTz during NWB0o, WB0o, and WB30o showed clear differences between the patients with PFP and healthy controls. In particular, the PF during WB30o(17.62o, extension) and the PTz during WB0o (72.50 mm, posterior) had the largest rotational and translational differences (JCS Δ = patients with PFP—controls), respectively. The JCS coordinates with statistically significant difference can serve as key biomarkers of patellar motion when evaluating a patient suspected of having PFP. The proposed method could reveal diagnostic biomarkers for accurately identifying PFP patients and be an effective addition to clinical diagnosis before surgery and to help plan rehabilitation strategies.